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One form of nucleotide excision repair (NER) is known to be functionally coupled to transcription, but the nature of this functional link in Escherichia coli is still unclear. Here we have employed the isolated membrane-associated nucleoids from E.coli to examine this issue. We show that the isolated nucleoid fraction is capable of excision of UV-induced pyrimidine dimers when reconstituted with a cytoplasmic fraction resolved by sucrose gradient fractionation. This excision activity by UvrABC is sensitive to rifampicin and is dependent on transcription. By using crosslinking and immunoprecipitation, the damage recognition protein, UvrA, was found to be specifically associated with the RNA polymerase beta subunit on the chromosomal DNA independent of DNA damage. It suggests that at least in one of the NER pathways the search for damage may be directly linked to RNA polymerase. In addition, the role of transcription in the unfolding of the nucleoid structure to allow repair enzymes to gain access to the damaged DNA is described. This study provides insight into the understanding of the transcription-repair coupling in vivo.

Transcription coupled nucleotide excision repair by isolated Escherichia coli membrane-associated nucleoids